Design and

purification of
proteins

Biotechnology project,
18/05/09

Marielle Brockhoff, Aurore Lacas , Raphael

Lieberherr Sebastian Olényi, Morgane
Perdomini, Zrinka Raguz,
Protein functions
ØTransport (O2)
ØRecognition (antibodies)
ØStructure/Architectur
e
ØCatalysis (enzymes)
ØCommunication
(hormon)
insulin production
Islet of Langerhans

ORGAN
ORGANIS TISS
M UE

FUNCTIONS

INFORMATION

DNA CELL (and

NUCLEUS)
genetic information of insulin
DNA ≈
Book

CHROMOSOME 11
≈
Chapter

Insulin ≈
GENE Sentence

CODON
≈
G Word
A T
G C
T 469 letters
..A CG AT
… .
from dna to insulin
Codon
- DNA
-
C GA T
- Insulin
Gl Va Gl Gl Cy
- y
Ile
l u n s

Protein = succession of
amino acids

Posttranslational
modifications
Hi

As
s Th
p r S
e
r
Th r
A
r g

Insulin correctly
folded
Protein structure
Primary structure

Secondary structure

Quaternary structure
Tertiary structure
 Insulin Structure
 469 letters 156 amino acids 51 amino
acids.
 two chains linked by disulfide bonds
 Insulin function

 Transportof
glucose requires
insulin

ØType 1 diabetes

ØType 2 diabetes

http://www.lillydiabetes.com/content/how-insulin-works.jsp
Protein Design
 Making entirely new or
modifying proteins for
example as drugs
Protein factories: From
bacteria to banana
Different advantages
Different modification
techniques
 Bacteria: viral transformation, artifical competence
(chemicals, electroporation)
 Plants: Agrobacterium, particle bombardment,
electroporation, viral transformation
 Humans, Animals: Chemistry, heat shock,
electroporation, viral transformation
Recombinant DNA
Technology in the Synthesis
 Since 1921: Treatement with
insulin derived from animals
 Bovine & porcine insulin
slightly different from
human insulin
 Sometimes inflammation at
injection sites
 Fear: long term
complications
 Solution: Inserting insulin
gene into E.coli to produce
identical human insulin
using Recombinant DNA
Technology
Manufacturing synthetic
human insulin
 Synthesis of the DNA containing the nucleotide
sequences of the A and B polypeptide chains of
insulin
Manufacturing synthetic
human insulin

Plasmid Plasmid + restriction

enzyme
 Insertion of the insulin
gene into plasmid
(circular DNA)
 Restriction enzymes cut
plasmidic DNA
 DNA ligase agglutinates
the insulin gene and the
plasmidic DNA
Plasmid + insulin gene
 Manufacturing synthetic
human insulin
 Introduction of recombinant
plasmids into bacteria: E. coli
 E.coli = factory for insulin
production
 Using E. coli mutants to avoid
insulin degradation
 Bacterium reproduces the
insulin gene replicates along
with plasmid

E. Coli
Manufacturing synthetic
human insulin
 Formed protein partly of a byproduct the A or B
chain of insulin
 Extraction and purification of A and B chains

byproduc byproduc

Insulin A-chain
Insulin B-chain
Manufacturing synthetic
human insulin
 Connection of A- and B-chain
 Reaction: Forming disulfide cross bridges
Result: Pure synthetic human insulin
Insulin production Today
 Yeast cells as growth medium
Secretion of almost complete human
insulin
Minimization of complex and
purification procedures

Yeast Insulin
Protein purification

Definition
Protein purification is a series of processes intended
to isolate a single type of protein from a complex
mixture of proteins
The applications of purified
proteins
Degree of purity
Depends on the application of the
protein!!!
 Industrial
applications: not so strict…
 Food and pharmaceuticals
 highlevel required, >99.99%
 Degree is set by the FDA (Food and Drug
Administration)
Properties of proteins used
for the purification
 Differences in proprieties allow a separation of
different proteins
 Properties come from
 Amino acids composition
 Amino adic chain length
 Structure/shape of the protein
(folding of the amino acid chain)
Properties of proteins
used for the purification
I. Size
Properties of proteins
used for the purification
I. Size

I. s
II. Charge

++ - +-- -
- ++ ++- - +-
++ + ++ + - - --
- - - -
+ +
+ o -
Properties of proteins
I. S
used for the purification
II. .
III. Solubility: pH, T, [Salt]

-+ -+ -+
-+

-+ + -+ -+
-+ Salt
I. S
Properties of proteins
II. .
used for the purification
III. .
IV. Hydrophobicity
I. S
Properties of proteins
II. .
used for the purification
III. .
IV. Hydrophobicity

I. S
II. .
III. .
IV. .

V. Specific binding
proprieties
Protein Purification
 Protein Location  Index
intracellular: - Filtration
sonication - Gel Filtration
extracellular - Ion Exchange
 Purification: chromatography
concentrate proteins, - Affinity
seperate proteins Chromatography
Filtration and
chromatography
Ultra Filtration
 Use: concentration,
desalting of proteins,
change buffer
 Membran: Pore size =

10-5 -10-2mm²
 Dialysis
Chromatography
 Purification using
specifique protein
properties, as: size,
charge,
hydrophobicity or
biorecognition
 Stationary phase:
inert material, or
coated material
 Mobile phase: buffer
Gel Filtration
 Mild conditions
(according to protein)
 With any buffer

 Isocratic

 Porous matrix in the

spherical beads
 Small proteins diffuse
into pores, stay
longer
Ion Exchange
Chromatography
 IEX
 Net surface charge

 According to pH and
the number and
exposure of amino
acids
 Charge = 0 at pI

 pH > pI protein –

 pH < pI protein +
Steps in IEX
 Matrix with bound
groups that are
charged
 Equilibration: adjust
pH in order that
protein of interest
binds to column
 Elution by changing
the ionic strength or
the pH
 Proteins with highest
charge elute latest
Affinity chromatography
 One step
 Specific binding
between protein and
ligand (eg substrate,
substrate analogue,
inhibitor, cofactor)
 His tag binds to metal
ions
Poly His Tag
 Commonly used for
recombinant proteins
 Ni2+ binds (His)6

 Eluting with imidazole

Insulin purification
 Extraction (separation of Bacteria/Yeasts)
 Purification (separation of other proteins) :

Cation exchange chromatography

OD measurement
 Precipitation with Zinc
Insulin extraction
 Secretion of insulin in medium: add sequence to
insulin gene
 Clarification of culture medium: isopropanol added
to medium, centrifugation and filtration

CENTRIFUGATION
Bacteria Medium with
insulin

Medium

get rid of Bacteria/Yeasts

Insulin Purification

 Ex: Cation exchange

Chromatography, SP Sepharose Fast
Flow
 Resin –CH2SO3-

 Total ionic capacity: 180-250μmol/ml gel

 Recommended flow rate: 100-300 cm/h
 Particle size range: 45-165 μm
 Working pH range: 4-13
 Maximum temperature: 30°C
Cation exchange Chromatography
 Resin Regeneration: 0.5N NaOH => resin is clean
 Equilibration: 20mM sodium citrate buffer at pH 4.0
=> fixation Na+
 Mix with insulin diluted with 20mM citrate buffer at
pH 4.0 => positively charged
 Loading of column and flow rate of 200cm/h =>
fixation of insulin

X
•CH2 REGENERATION Na+ +
SO3-
•CH2 ADD MIX •CH2
SO3- SO3-
Y EQUILIBRATION
Na+ insulin + +

resin
Cation exchange Chromatography
 Washing: 20mM citrate buffer => elimination of
molecules not fixed
 Elution: 100mM tris HCl, pH 7.5 buffer, flow rate of
100cm/h => replacement of insulin by H+

+
•CH2 + ELUTION +H
•CH2 •CH2
SO3-
SO3- SO3-
+ Low HCl
concentration + +H

Fraction
with buffer
and no
insulin
Fraction with
insulin
Determination of fractions
containing insuline
 OD 280nm
Aromatic amino acid absorb at 280nm =>
detection of protein presence in solution
 A= εlC ε280nm=0.55 x 104 M-1cm-1

Phenylalanin Tryptophan Tyrosin

Precipitation with Zinc

 Add ZnCl2 to purified insulin and adjust pH to

6 => precipitation
 Refrigerator (8 °C) for at least 6h

 Centrifugation 5000rpm

 Drying of pellet => dry insulin

 Yield for ion exchange chromatography and

precipitation: around 75%
 CONCLUSION
 Productionof proteins is a big market
Example: Lilly Insulin production
since 1923

 Nessecity of good design and

purification protocol
Thank you for your
attention

Questions?